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Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.

1. To harvest the Nematocida culture, add 5 ml of M9 buffer to an infected NGM plate and transfer the suspension to a 15 ml centrifuge tube.

2. Centrifuge at 200 x g for 5 min. Remove the supernatant, resuspend the pellet in 5 ml of M9 buffer, and repeat the centrifugation step.

3. Repeat step 2 a minimum of five times in order to wash the infected worms.

4. After the last wash, resupend the pellet in 1 ml of M9 buffer and transfer the suspension to a 2 ml microcentrifuge tube. Add Silicon carbide beads (BioSpec Products, Inc.) to the tube and vortex for 1 minute. Repeat the procedure 4-5 times.

4. Filter the worm extract through a Whatman filter paper number 1 to remove eggs and any remaining intact worms.

5. Perform a spore count of the worm extract and adjust the concentration to ≥ 3 x 107 spores/ml. NOTE: If the concentration of spores is too low, harvest infected worms from additional NGM plates to yield the desired concentration.

6. Mix the extract with an equal volume of M9 buffer containing 30% glycerol. The final concentration of the extract will be ≥ 1.5 x 107 spores/ml and 15% glycerol.

8. Place vials in a controlled rate freezing unit. From room temperature cool at -1°C/min to -40°C. If freezing unit can compensate for the heat of fusion, maintain rate at -1o C/min through heat of fusion. At -40°C plunge ampules into liquid nitrogen. Alternatively, place the vials in a Nalgene 1°C freezing apparatus. Place the apparatus at -80°C for 1.5 to 2 hours and then plunge ampules into liquid nitrogen. (The cooling rate in this apparatus is approximately

-1°C/min.)

9. Store frozen ampules in either the vapor or liquid phase of a nitrogen refrigerator.

Customers located in the state of Hawaii will need to contact the Hawaii Department of Agriculture to determine if an Import Permit is required. A copy of the permit or documentation that a permit is not required must be sent to ATCC in advance of shipment.